Project Summary/Abstract The premise of this proposal is that there is a dearth of biochemical evidence for the current dogma of a 3 layered tear film model. The treatment of dry eye disease requires knowledge of the distribution and interactions of biologic components that comprise tear film structure. Currently, the scientific basis for treatment is lacking. The strategy of replacement of absent component or bolstering components that function in a certain way has been the basis for ingredients for dry eye and it has not worked well. This approach is flawed until locations and functions of components are determined. The need for better treatment can not be met until normal tear film structure is understood. The distribution of lipids and proteins tears has been presumed from non-specific interferometry data without biochemical proof. A major obstacle to understanding the distribution of the components has been the lack of technology to track individual molecules and their functional interactions. The lipid layer of tear film is tens of nanometers in thickness. Direct visualization of dynamic components in solution was until now beyond the reach of scientists. This project will capitalize on recent technological advances in fluorescence microscopy and fluorescence spectroscopy to track lipids and proteins. Two proposal aims are designed to show the distribution of the major components of lipids and proteins in the tear film. The first aim will use customized sub-diffraction (super-resolution)microscope to localize fluorophores in the compartments of tears at a resolution of 9 nm. The microscope was specifically constructed for tear film structure. The second aim proposes to incorporate highly sensitive single photon counting fluorescence measurements to resolve interactions of individual molecules at sub-nanometer distances. Our laboratory has honed the facilities, equipment, reagents and technical expertise to make these measurements. Success with this proposal will lead to biochemical data for the distribution of major lipids and proteins that comprise tears. The research may replace models with a scientific foundation to test functions of the tear film components and create solutions for dry eye diseases.